Iron-iron hydrogenases are highly active hydrogen-producing enzymes. Hydrogenases mediate molecular processes of broad biological and medical significance: the evolution and transport of gases and the transfer of electrons through FeS clusters. Hydrogenases lie at the heart of the metabolism of several anaerobic parasites, including the human parasite Trichomonas vaginalis. The coupling of hydrogenases to photosynthesis offers the possibility of the clean production of H2 by engineered microorganisms. However, the connection of photosynthesis to hydrogenases is complicated by the sensitivity of the active sites of hydrogenases to oxygen. The specific aims of this proposal are: Aim 1: Directed Evolution of O2-tolerant Hydrogenases using Genetic Screens; Aim 2: Construction, Characterization and Evolution of a Library of Novel FeFe-hydrogenases; Aim 3: Characterization and Evolution of Hydrogenase Accessory Proteins